The degradation of native and 125I-labeled human insulin (HI) was examined in the cytosolic fraction of human, monkey, and rat liver. The purpose of these studies was to provide a species comparison of the interaction of insulin-degrading enzyme (IDE) and protein disulfide isomerase (PDI) in the degradation of HI. Western-blot analysis with monoclonal antibodies indicated the presence of both IDE and PDI in the cytosolic fraction of human and monkey liver. In contrast, rat liver cytosol contained, detectable levels of IDE only. A species comparison of metabolic profiles was performed by fractionating peptide products with reversed-phase high-performance liquid chromatography. After a 60-min incubation, human liver cytosol degraded unlabeled HI into three major products. Two of these peptides coeluted with the products of the incubation of HI with purified rat liver PDI. The three peptides were isolated and determined by NH2-terminal sequence analysis to be intact A chain, B chain, and des(Phe1)–B chain. Human liver cytosol also formed 125I–A chain and 125I–B chain as major products when specifically labeled 125I–HI isomers were used as substrate. Significant proteolytic degradation was observed only when reactions with human liver cytosol were supplemented with Mn2+. In contrast, monkey and rat liver cytosol proteolytically degraded 125I–HI isomers to small peptide fragments. The rat and monkey metabolic profiles were similar to each other and to that observed with Mn2+-supplemented human liver cytosol. Proteolysis in monkey and rat was sensitive to inhibition by EDTA. When proteolysis was inhibited in monkey cytosol, the formation of B chain, reflecting the activity of PDI, was evident. Immunoinhibition of PDI with ascites containing monoclonal antibodies to PDI prevented the metabolism of 125I-(B26)HI in monkey and Mn2+ supplemented human liver cytosol. PDI antibodies did not inhibit the proteolysis of 125I-(B26)-HI in rat cytosol or the proteolysis of 125I-(B26)–B chain by monkey or Mn2+-supplemented human liver cytosol. These results are consistent with the involvement of both PDI and IDE in the degradation of HI by nonrodent and human liver cytosol.

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